Chemokines are a group of inflammatory/immunomodulatory polypeptide factors produced by lymphatic tissues and by activated macrophages and leukocytes at inflammatory sites; they have a molecular weight of 6–15 kD, contain four cysteine residues, are basic and have heparin binding activity. The chemokines can be classified into two subfamilies, the CXC chemokines and CC chemokines, by the common location of the four cysteine residues and by the differences in the chromosomal locations of the genes encoding them. For example IL-8 (abbreviation for interleukin-8) is a CXC chemokine, while the CC chemokines include MIP-1α/β (abbreviation for macrophage inflammatory protein-1α/β), MCP-1 (abbreviation for monocyte chemotactic protein-1), and RANTES (abbreviation for regulated on activation, normal T-cell expressed and secreted cytokine). There also exists a chemokine called lymphotactin, which does not fall into either chemokine subfamily. These chemokines promote cell migration, increase the expression of cellular adhesion molecules such as integrins, and promote cellular adhesion, and are thought to be the protein factors involved in the adhesion and infiltration of leukocytes into the pathogenic sites in inflammatory tissues (for references, see for example, Michiel, D., Biotechnology, 1993, 11, 739; Oppenheim, J. J., et al., Annual Review of Immunology, 1991, 9, 617–648; Schall, T. J., Cytokine, 1991, 3, 165–183; Springer, T. A., Cell, 1994, 76, 301–314; Furie, M. B., American Journal of Pathology, 1995, 146, 1287–1301; Kelner, G. S., et al., Science, 1994, 266, 1395–1399).
For example, MIP-1α induces cell migration and causes a transient increase in intracellular calcium ion concentration levels, an increase in the expression of integrins, and adhesion molecules, and degranulation of monocytes and lymphocytes, and inhibits bone marrow stem cell proliferation (See for example, Wolpe, S. D., et al., Journal of Experimental Medicine, 1988, 167, 570–581; Wolpe, S. D., et al., Faseb Journal, 1989, 3, 2565–2573; Taub, D. D., et al., Science, 1993, 260, 355–358; Schall, T. J., et al., Journal of Experimental Medicine, 1993, 177, 1821–1825; Neote, K., et al., Cell, 1993, 72, 415–425; Vaddi, K., et al.; The Journal of Immunology, 1994, 153, 4721–4732).
With respect to the activity of MIP-1α in vivo and its role in the pathogenesis of disease, it has been reported that it is a pyrogen in rabbits (see for example Davatelis, G., et al., Science, 1989, 243, 1066–1068); that MIP-1α injection into mouse foot pads results in an inflammatory reaction such as infiltration by neutrophils and mononuclear cells (see for example Alam, R., et al., The Journal of Immunology, 1994, 152, 1298–1303); that MIP-1α neutralizing antibody has an inhibitory effect or a therapeutic effect in animal models of granuloma, multiple sclerosis and idiopathic pulmonary fibrosis (see for example Lukacs, N. W., et al., Journal of Experimental Medicine, 1993, 177, 1551–1559; Karpus, W. J., et al., The Journal of Immunology, 1995, 155, 5003–5010; Smith, R. E., et al., The Journal of Immunology, 1994, 153, 4704–4712); and that coxsackie virus induced myocarditis is inhibited in mice with a disrupted MIP-1α gene (see for example Cook, D. N. et al., Science, 1995, 269, 1583–1585). These studies indicate that MIP-1 α is involved in the local attraction of various subtypes of leukocytes and the initiation, progression and maintenance of resulting inflammatory response.
These data indicate that chemokines, such as MIP-1α, attract monocytes and lymphocytes to disease sites and mediate their activation, and thus are involved in the initiation, progression and maintenance of diseases pertaining to monocytes and lymphocytes, such as atherosclerosis, rheumatoid arthritis, transplant rejection, psoriasis, asthma, ulcerative colitis, glomerulonephritis, multiple sclerosis, pulmonary fibrosis and myocarditis.
Therefore, drugs that inhibit the action of chemokines on target cells are effective as therapeutic and/or preventive drugs in diseases such as atherosclerosis, rheumatoid arthritis, transplant rejection, psoriasis, asthma, ulcerative colitis, glomerulonephritis, multiple sclerosis, pulmonary fibrosis, and myocarditis.
The chemokines bind specific cell-surface receptors belonging to the family of G-protein-coupled seven-transmembrane-domain proteins (reviewed in Horuk, Trends Pharm. Sci., 15, 159–165 (1994)), which are termed “chemokine receptors.” On binding their cognate ligands, chemokine receptors transduce an intracellular signal through the associated trimeric G protein, resulting in a rapid increase in intracellular calcium concentration. There are at least seven human chemokine receptors that bind or respond to CC-chemokines with the following characteristic pattern: CCR-1 (or “CKR-1” or “CC-CKR-1”) [MIP-1α, MIP-β, MCP-3, RANTES] (Ben-Barroch, et al., J. Biol. Chem., 270, 22123–22128 (1995), Beole, et al, Cell, 72, 416–426 (1993)); CCR-2A and CCR-2B (or “CKR-2A”/“CKR-2A” or “CC-CKR-2A:/:CC-CKR-2A”) [MCP-1, MCP-3, MCP-4]; CCR-3 (or “CKR-3: or CC-CKR-3”) [eotaxin, RANTES, MCP-3] (Combadiere, et at., J. Biol. Chem., 270, 16491–16494 (1995); CCR-4 (or “CKR-4” or “CC-CKR-4”) [MIP-1α, RANTES, MCP-1] (Power, et at., J. Biol. Chem., 270, 19495–19500 (1995); CCR-5 (or “CKR-5” or “CC-CKR-5”) [MIP-1α, RANTES, MIP-1β] (Sanson, et al., Biochemistry, 35, 3362–3367 (1996); and the Duffy blood-group antigen [RANTES, MCP-1] (Chaudhun, et al., J. Biol. Chem. 269, 7835–7838 (1994)).
Chemokine receptors, such as CCR-1, CCR-2, CCR-2A, CCR-2B, CCR-3, CCR-4, CCR-5, CXCR-3, CXCR-4, are considered important mediators of inflammatory and immunoregulatory disorders and diseases, including asthma and allergic diseases, as well as autoimmune pathologies such as rheumatoid arthritis and atherosclerosis. Accordingly, agents which modulate chemokine receptors would be useful in such disorders and diseases.
A retrovirus designated human immunodeficiency virus (HIV-1) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV.
Certain compounds have been demonstrated to inhibit the replication of HIV, including soluble CD4 protein and synthetic derivatives (Smith, et al., Science, 238, 1704–1707 (1987)), dextran sulfate, the dyes Direct Yellow 50, Evans Blue, and certain azo dyes (U.S. Pat. No. 5,468,469). Some of these antiviral agents have been shown to act by blocking the binding of gp120, the coat protein of HIV, to its target, the CD4 glycoprotein of the cell.
Entry of HIV-1 into a target cell requires cell-surface CD4 and additional host cell cofactors. Fusin has been identified as a cofactor required for infection with virus adapted for growth in transformed T-cells, however, fusin does not promote entry of macrophage-tropic viruses, which are believed to be the key pathogenic strains of HIV in vivo. It has recently been recognized that for efficient entry into target cells, human immunodeficiency viruses require the chemokine receptors CCR-5 and CXCR-4, as well as the primary receptor CD4 (Levy, N. Engl. J. Med., 335(20), 1528–1530, (Nov. 14, 1996)). The principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-trophic strains of HIV-1 is CCR5, a receptor for the β-chemokines RANTES, MIP-1α and MIP-1β (Deng, et al., Nature, 381, 661–666 (1996)). HIV attaches to the CD4 molecule on cells through a region of its envelope protein, gp120. It is believed that the CD4 binding site on the gp120 of HIV interacts with the CD4 molecule on the cell surface, and undergoes conformational changes, which allow it to bind to another cell-surface receptor, such as CCR5 and/or CXCR-4. This brings the viral envelope closer to the cell surface and allows interaction between gp41 on the viral envelope and a fusion domain on the cell surface, fusion with the cell membrane, and entry of the viral core into the cell. It has been shown that β-chemokine ligands prevent HIV-1 from fusing with the cell (Dragic, et al., Nature 381, 667–673 (1996)). It has further been demonstrated that a complex of gp120 and soluble CD4 interacts specifically with CCR-5 and inhibits the binding of the natural CCR-5 ligands MIP-1 α and MIP-1β (Wu, et al., Nature 384, 179–183 (1996); Trkola, et al., Nature, 384, 184–187 (1996)).
Humans who are homozygous for mutant CCR-5 receptors, which do not serve as co-receptors for HIV-1 in vitro, appear to be unusually resistant to HIV-1 infection and are not immunocompromised by the presence of this genetic variant (Nature, 332, 722–725 (1996)). Absence of CCR-5 appears to confer protection from HIV-1 infection (Nature, 382, 668–669 (1996)). Other chemokine receptors may be used by some strains of HIV-1 or may be favored by non-sexual routes of transmission. Although most HIV-1 isolates studied to date utilize CCR-5 or fusin, some can use both as well as the related CCR-2B and CCR-3 as co-receptors (Nature Medicine, 2(11), 1240–1243 (1996)). Nevertheless, drugs targeting chemokine receptors may not be unduly compromised by the genetic diversity of HIV-1 (Zhang, et al., Nature, 383, 766 (1996)). Accordingly, an agent that blocks chemokine receptors in humans who possess normal chemokine receptors should prevent infection in healthy individuals and slow or halt viral progression in infected patients. By focusing on the host's cellular immune response to HIV infection, better therapies towards all subtypes of HIV are provided. These results indicate that inhibition of chemokine receptors presents a method for the prevention or treatment of infection by HIV and the prevention or treatment of AIDS.
The peptides eotaxin, RANTES, MIP-1 α, MIP-1β, MCP-1, and MCP-3 are known to bind to chemokine receptors. As noted above, the inhibitors of HIV-1 replication present in supernatants of CD8+T cells have been characterized as the β-chemokines RANTES, MIP-1α and MIP-1β. Therefore, compounds that inhibit the binding of chemokines such as MIP-1α to these receptors, that is, chemokine receptor antagonists, are useful as drug targets that inhibit the action of chemokines on the target cells.
Recently, it was reported that the diphenylmethane derivatives (WO9724325) and other small molecules (WO9744329; WO9802151; WO9804554) are antagonists of chemokine receptors, such as the MIP-1α/RANTES receptor (defined as CCR1).